Gps2, a protein partner for human papillomavirus E6 proteins - PubMed (original) (raw)

Gps2, a protein partner for human papillomavirus E6 proteins

Y Y Degenhardt et al. J Virol. 2001 Jan.

Abstract

We have used the yeast two-hybrid system to screen a cDNA library prepared from normal human epidermal keratinocytes and identified protein partners for human papilloma virus (HPV) E6 proteins. A clone that encoded Gps2 interacted with E6 proteins from HPVs of high and low oncogenic risk. The specificity of these reactions was verified and the regions of E6 that were required for interaction were mapped. Steady-state and pulse-chase analyses of cells cotransfected with DNAs expressing E6 from either HPV6 or HPV18 and Gps2 demonstrated that the E6 proteins induced the degradation of Gps2 in vivo but not in vitro. Gps2 exhibited transcriptional activation activity, and high-risk E6 suppressed this activity.

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Figures

FIG. 1

FIG. 1

E6 proteins binding to Gps2. (A) In vitro-translated and 35S-labeled 6E6, 18E6, and the negative control LIM domain from zyxin were incubated with GST or GST-Gps2 fusion proteins bound to agarose beads. E6 proteins that interacted with the bound baits were eluted from the beads and subjected to SDS-PAGE analysis followed by autoradiography. (B) In vitro-translated and 35S-labeled Gps2 was allowed to interact with equal amounts of purified GST, GST-E6, or GST-p300 fusion proteins. After binding at 50 mM salt, the agarose beads were subjected to washes with buffer containing increasing concentrations of salt (millimolar) as indicated above the lanes. After extensive washes, the bound Gps2 was eluted from the beads and subjected to SDS-PAGE analysis followed by autoradiography. The lanes marked input contain 10% of the reacting material.

FIG. 2

FIG. 2

Coimmunoprecipitation of E6 with Gps2. (A) 293T cells were transfected with 20 μg of the Flag vector alone (lanes 1, 4, and 7), Gps2-Flag (lanes 2, 5, and 8), or PKC-Flag (lanes 3, 6, and 9). Cell lysates were prepared 36 h after transfection. Equal amounts of total protein were mixed with 10 μl of in vitro-translated, 35S-labeled 6E6, 18E6, or LIM and then immunoprecipitated with anti-Flag antibody. Coprecipitated proteins were subjected to SDS-PAGE and analyzed by autoradiography. Lanes 10, 11, and 12 contain 10% of the 6E6, LIM, or 18E6 input material, respectively. (B) After immunoprecipitation with anti-Flag antibody, Sepharose beads were boiled in SDS loading buffer and subjected to SDS-PAGE analysis. The retention of PKC-Flag and Gps2-Flag proteins was monitored by Western blot analysis with an anti-Flag antibody. (C) 293T cells were transfected with a plasmid expressing Gsp2-Flag, Myc-tagged E6, or both plasmids. At 36 h after transfection, cells were labeled with 35S-Translabel for 3 h, and then whole-cell lysates were prepared. Equal amounts of protein from each lysate were subjected to immunoprecipitation with anti-Flag affinity gel, and the coprecipitated proteins were subjected to SDS-PAGE and autoradiography. The bands corresponding to E6 and Gps2 are indicated alongside the gel. (D) E6 expression in each of the above cotransfections was measured by Western analysis using an anti-Myc antibody.

FIG. 3

FIG. 3

Interaction between cutaneous HPV E6 and Gps2. (A) HPV E6–Gal4-AD fusions were cotransformed with a Gps2-LexA fusion or the LexA vector into yeast strain L40. Liquid β-gal assays were performed on the cotransformed colonies as described in Materials and Methods. The β-gal values are averages of three separate experiments. (B) In vitro-translated and 35S-labeled Gps2 was allowed to interact with equal amounts of purified GST or GST-E6 fusion proteins bound to agarose beads. Captured Gps2 was assayed by SDS-PAGE and autoradiography after boiling the beads in SDS loading buffer. (C) The amount of GST fusion protein used in each binding assay was determined by Coomassie brilliant blue staining of the SDS-PAGE gel shown in panel B. Interaction with GST-p300 was assayed as an additional negative control.

FIG. 4

FIG. 4

Identification of Gps2-interactive domains in E6. (A) Schematic representation of the 6E6 ORF. Deletions of 6E6 were fused in frame to Gal4-AD in the yeast vector pACTII for Gps2 binding studies in yeast cells. The numbers of the starting and ending amino acids in 6E6 define the deletion mutants. (B) Yeast strain L40 was cotransformed with Gal4-AD fusions of 6E6 deletions and Gps2-LexA. Liquid β-gal assays were performed on the contransformants as described in Materials and Methods. The values are averages from three separate experiments.

FIG. 5

FIG. 5

Interaction of truncated HPV E6 proteins with GST-Gps2. In vitro-translated and 35S-labeled 6E6 proteins containing the amino acids specified above the lanes and LIM were allowed to interact with GST-Gps2. The bound proteins were separated by SDS-PAGE and visualized by autoradiography. The percentage of input material bound is indicated below the lanes. ND, not detectable.

FIG. 6

FIG. 6

Degradation of Gps2 in vivo. Gps2-Flag construct (250 ng) was cotransfected with 2.5 μg of 6E6-HA, 18E6-HA, or HA vector pHANE and 50 ng of a CMV-Luc reporter into NIH 3T3 cells. The lane labeled Mock contained only the Flag and HA vector DNAs. (A) Thirty-six hours posttransfection, cell lysates were collected, and equal amounts of total protein were subjected to SDS-PAGE and Western blot analysis using an anti-Flag antibody to detect the protein level of Gps2. The arrow points to the bands corresponding to Gps2. The normalized intensities of the Gps2 bands, as displayed below the lanes, were determined by dividing the absolute intensity of the respective bands by the level of luciferase expressed by the cotransfected reporter control. (B) The levels of E6 expressed by the 6E6-HA and 18E6-HA constructs were assayed by probing a Western blot prepared using extracts from the experiment described above with anti-HA antibody. (C) Cultures of cotransfected NIH 3T3 cells (without the CMV-Luc internal control) were pulse-labeled and chased as described in Materials and Methods, and cell lysates were prepared directly after the pulse (time zero) and after 1 and 3 h. The lysates with the same amounts of protein were immunoprecipitated with an anti-Flag antibody and subjected to SDS-PAGE, and the labeled Gps2 was visualized and quantified with a PhosphorImager. The percentage of Gps2 remaining at each time point is presented below the lanes. ND, not detectable.

FIG. 7

FIG. 7

Assay for degradation of Gps2 in vitro. In vitro-translated and 35S-labeled Gps2 or p53 was mixed with in vitro-translated HPV E6 proteins and analyzed for degradation as described in Materials and Methods. An unprimed reticulocyte lysate and in vitro-translated herpes simplex virus ICP27 protein were used as negative controls. One-fifth of the total reaction was subjected to SDS-PAGE, and the bands for Gps2 and p53 were visualized using a PhosphorImager.

FIG. 8

FIG. 8

Transactivation by Gps2 and the effect of E6. Luciferase reporters driven by the indicated promoters were transfected into NIH 3T3 cells with or without Gps2 or HPV E6s. The amount of DNA for the luciferase reporter, Gps2, and E6 in the transfections was 100, 500, and 1,500 ng, respectively. The vectors for Gps2 and HPV E6 constructs were also added to keep the total amount of DNA in each transfection constant. RL-TK DNA was added to each transfection as a control for transfection efficiency. Cell lysates were collected at 36 h after transfection, and the firefly and Renilla luciferase activities were quantitated as described in Materials and Methods. The ratio of firefly luciferase to Renilla luciferase in cells transfected with only these constructs was set at 1. For each of the other transfections, the ratio of the firefly luciferase to Renilla luciferase was normalized to the ratio described above and plotted as relative luciferase units. Each point shown is the average of two separate experiments.

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